Important amino acid residues of hexachlorocyclohexane dehydrochlorinases (LinA) for enantioselective transformation of hexachlorocyclohexane isomers

LinA-type1 and LinA-type2 are two well-characterized variants of the enzyme ‘hexachlorocyclohexane (HCH)-dehydrochlorinase’. They differ from each other at ten amino acid positions and exhibit differing enantioselectivity for the transformation of the (–) and (+) enantiomers of α-HCH. Amino acids responsible for this enantioselectivity, however, are not known. An in silico docking analysis identified four amino acids (K20, L96, A131, and T133) in LinA-type1 that could be involved in selective binding of the substrates. Experimental studies with constructed mutant enzymes revealed that a combined presence of three amino acid changes in LinA-type1, i.e. K20Q, L96C, and A131G, caused a reversal in its preference from the (–) to the (+) enantiomer of α-HCH. This preference was enhanced by the additional amino acid change T133 M. Presence of these four changes also caused the reversal of enantioselectivity of LinA-type1 for δ-HCH, and β-, γ-, and δ-pentachlorocyclohexens. Thus, the residues K20, L96, A131, and T133 in LinA-type1 and the residues Q20, C96, G131, and M133 in LinA-type 2 appear to be important determinants for the enantioselectivity of LinA enzymes.     

Metabolomics of hexachlorocyclohexane (HCH) transformation: ratio of LinA to LinB determines metabolic fate of HCH isomers

Although the production and use of technical hexachlorocyclohexane (HCH) and lindane (the purified insecticidal isomer γ‐HCH) are prohibited in most countries, residual concentrations still constitute an immense environmental burden. Many studies describe the mineralization of γ‐HCH by bacterial strains under aerobic conditions. However, the metabolic fate of the other HCH isomers is not well known. In this study, we investigated the transformation of α‐, β‐, γ‐, δ‐, ε‐HCH, and a heptachlorocyclohexane isomer in the presence of varying ratios of the two enzymes that initiate γ‐HCH degradation, a dehydrochlorinase (LinA) and a haloalkane dehalogenase (LinB). Each substrate yielded a unique metabolic profile that was strongly dependent on the enzyme ratio. Comparison of these results to those of in vivo experiments with different bacterial isolates showed that HCH transformation in the tested strains was highly optimized towards productive metabolism of γ‐HCH and that under these conditions other HCH‐isomers were metabolized to mixtures of dehydrochlorinated and hydroxylated side‐products. In view of these results, bioremediation efforts need very careful planning and toxicities of accumulating metabolites need to be evaluated.     

Stereospecificity in the cytotoxic action of hexachlorocyclohexane isomers

Hexachlorocyclohexane (HCH) is a highly recalcitrant organochlorine insecticide known for its chronic toxicity. In spite of many isolated studies a clear mechanism of cytotoxic action of HCH and the structure–toxicity relationship of its isomers is not well understood. We have investigated the toxicity of HCH isomers and its mechanism in Ehrlich Ascites tumor (EAT) cells. Our studies show differential cytotoxicity of HCH isomers (α, β, γ, and δ), δ isomer being most toxic and β the least. HCH-induced cell death was associated with induction of reactive oxygen species (ROS) formation, lipid peroxidation (LPO), and depletion of glutathione (GSH). The increase in oxidative stress was linked with increased NAD(P)H oxidase activity. HCH inhibited Na⁺,K⁺-ATPase, which could be involved in raising the intracellular calcium and increased Ca²⁺,Mg²⁺-ATPase activity. HCH lead to apoptotic as well as necrotic cell death as it was marked by increased caspase-3 activity and lactate dehydrogenase (LDH) leakage, respectively. Based on the results it is concluded that the HCH isomers inflict differential cytotoxicity which was highest by δ and lowest by β. Further, this study demonstrates for the first time a clear link between Na⁺,K⁺-ATPase, i[Ca²⁺] level, and oxidative stress in HCH-induced cytotoxicity.     

Influence of dietary hexachlorocyclohexane isomers on lipid metabolism in albino rats

Dietary intake of beta- and gamma-hexachlorocyclohexane (beta- and gamma-HCH) by albino rats for two weeks (at 800 ppm level) resulted in impairment of lipid metabolism, viz. hyperlipemia and fatty metamorphosis of liver. Liver fat content was increased by both beta- and gamma-HCH. Significant increases were observed in triglyceride and phospholipid fractions of blood in these experimental animals. The incorporation of [14C]acetate and palmitate into liver and blood lipids was higher in HCH pretreated animals, suggesting a higher rate of fat synthesis in liver and of secretion as well.     

Anaerobic dechlorination and degradation of hexachlorocyclohexane isomers by anaerobic and facultative anaerobic bacteria

Screening studies with strict and facultative anaerobic bacteria showed that Clostridium app. and several other representatives of Bacillaceae and Enterobacteriaceae actively degraded -hexachlorocyclohexane (-HCH) under anaerobic conditions. Representatives of Lactobacillaceae and Propronibacterium were inactive. With ³⁶Cl-labelled -HCH a nearly complete dechlorination was shown to occur in 4–6 days by Clostridium butyricum, C. pasteurianum and Citrobacter freundii, while other facultative anaerobic species were less active.Aerobically grown facultative anaerobes also dechlorinated actively -HCH during subsequent anaerobic incubation with glucose, pyruvate or formate as substrates. The -, - and -HCH isomers were also, but more slowly, dechlorinated (>>-HCH). All species active in anaerobic degradation of -HCH formed -tetrachlorocyclohexene (TCH) as the main intermediate metabolite and no -pentachlorocyclohexene (PCH) or other isomers of TCH or PCH have been found. Small amounts of tri- and tetrachlorinated benzenes have been found too. The mechanism of dechlorination is discussed.Non-Common Abbreviations Used -HCH -hexachlorocyclohexane - -TCH -2,3,4,5-tetrachlorocyclohexene - -PCH -1,2,3,4,5-pentachlorocyclohexene - GLC gas liquid chromatography     

A database analysis of jacalin-like lectins: sequence-structure-function relationships

Lectins are known to be important for many biological processes, due to their ability to recognize cell surface carbohydrates with high specificity. Plant lectins have been model systems to study protein-carbohydrate recognition, because individually they exhibit high sensitivity and as a group large diversity in recognizing carbohydrate structures. Although extensive studies have been carried out for legume lectins that have led to interesting insights into the sequence determinants of sugar recognition in them, frameworks with such specific correlations are not available for other plant lectin families. This study reports a large-scale data acquisition and extensive analysis of sequences and structures of beta-prism-I or jacalin-related lectins (JRLs) and shows that hypervariability in the binding site loops generates carbohydrate recognition diversity, a strategy analogous to that in legume lectins. Analyses of the size, conformation, and sequence variability in key regions reveal the existence of a common theme, encoded as a set of structural features over a common scaffold, in defining specificity. This study also points to the remarkable range of domain architectures, often arising out of gene duplication events in lectins of this family. The data analyzed here also indicate a spectacular variety of quaternary associations possible in this family of lectins that have implications for glycan recognition. These results thus provide sequence-structure-function correlations, an understanding of the molecular basis of carbohydrate recognition by beta-prism-I lectins, and also a rationale for engineering specific recognition capabilities in relevant molecules.     

Effects of hexachlorocyclohexane isomers on concanavalin A 'capping' in bovine lymphocytes.

The effects of alpha, beta, gamma, and delta isomers of hexachlorocyclohexane on concanavalin A 'capping' in bovine lymphocytes were evaluated, gamma and delta hexochlorocyclohexane inhibited 'capping' whereas the alpha and beta isomers were without effect. In addition, gamma-hexachlorocyclohexane has been shown to antagonize the maintenance of preformed 'caps' and cause the rapid dispersal of the concanavalin A-receptor complexes over the surface of cells by a temperature-dependent mechanism. The possible role of a gamma-hexachlorocyclohexane-sensitive process in the organization of microflow patterns in the lectin-activated lymphocyte membrane is discussed.     

Degradation by and toxicity to bacteria of chlorinated phenols and benzenes,and hexachlorocyclohexane isomers

Mixed cultures degrading chlorinated benzenes, chlorinated phenols, or hexachlorocyclohexane (HCH) as the sole source of carbon and energy were obtained by enrichment from contaminated soil samples. Cultures which metabolized 3-chlorophenol (3-CP), 2,3-dichlorophenol (2,3-DCP), or 2,6-dichlorophenol (2,6-DCP) were able to utilize several other chlorinated compounds as substrates, whereas cultures enriched with 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB), -HCH, or -HCH did not metabolize most of the other chlorinated congeners tested. Chloride release and growth rates with all four chlorinated phenols decreased with increasing initial substrate concentrations within the range of 30–250 mol liter^–1. Maximum chloride release was 3.8 mg liter^–1 corresponding to 35 mol liter^–1 trichlorophenol within 7 weeks. In contrast, the rate of metabolism of the nonphenolic compounds 1,2,4,5-TeCB, -HCH, or -HCH increased with increasing substrate concentrations. Initial concentrations of 750 mol liter^–1 -HCH or 1,2,4,5-TeCB were completely dechlorinated within 2 weeks. Because aqueous solubility and bioavailability of the chlorophenolic compounds is much higher than that of the nonphenolic compounds, it is suggested that the high bioavailability of the chlorophenolic compounds is the reason for the high toxicity of these substrates to the degrading cultures. In contrast, the low aqueous solubilities of the chlorinated benzenes and HCH-isomers caused consistently low concentrations in the medium, which were high enough to induce degradation but too low to damage the bacterial cells.Correspondence to: E. Lang     

Differential presynaptic effects of hexachlorocyclohexane isomers on noradrenaline release in cerebral cortex.

To investigate presynaptic effects of hexachlorocyclohexane (HCH) isomers, the release of noradrenaline (NA) in brain tissue was analyzed using rat cerebral cortical slices preloaded with [3H]-NA. gamma-HCH (lindane) 50 microM significantly enhanced the [3H]-NA release evoked by 15-25 mM K+. alpha- and beta-HCH (50 microM) did not produce any significant effect on K(+)-evoked [3H]-NA release. delta-HCH (50 microM) induced a significant decrease of the 25 mM K(+)-evoked release of [3H]-NA. The effect of the gamma- and delta-HCH isomers on the presynaptic action of the alpha 2-agonist clonidine and the alpha 2-antagonist yohimbine was also studied. The presynaptic inhibitory effect of clonidine and the stimulatory effect of yohimbine on [3H]-NA release was attenuated by lindane and delta-HCH, respectively. These results are consistent with a presynaptic action of the HCH isomers on noradrenergic release processes.     

Characterization of phosphoinositide hydrolysis products induced by hexachlorocyclohexane isomers in rat brain cortex

Water-soluble inositol metabolites were separated by anion-exchange chromatogrphy in order to determine whether or not -hexachlorocyclohexane (-HCH, lindane) and related compounds affect phosphatidylinositol hydrolysis in rat brain cortex slices. Hydrolysis was increased by -and -HCH, while - and -HCH were inactive. Muscarinic receptor stimulation of rat cortical slices with carbachol increases inositol phosphates formation. The combined effect of carbachol and the hexachlorocyclohexane isomers together were approximately equal to the sum of the effect of each one separately. The results suggest that lindane stimulates phosphoinositide phospholipase C and/or inhibits the phosphases implicated in dephosphorylation of inositol phosphates.     

Reductive dechlorination of hexachlorocyclohexane (HCH) isomers in soil under anaerobic conditions

The biological anaerobic reductive dechlorination of beta-hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of beta-hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of beta-hexachlorocyclohexane within 4 months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (alpha-, beta-, gamma-, and delta-), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30 degrees C. Dechlorination of beta-hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10 mM sulphate in the influent led to simultaneous dechlorination of beta-hexachlorocyclohexane and sulphate reduction. When the column was fed with 10 mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1 month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of beta-HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed.     

Opposite effects of different hexachlorocyclohexane (lindane) isomers on cerebellar cyclic GMP: relation of cyclic GMP accumulation to seizure activity

A number of different depressant and convulsant agents have been shown to alter accumulation of cerebellar cyclic GMP. Since the different hexachlorocyclohexane (HCH) isomers elicit different pharmacological responses in mammals, we examined their effects on the accumulation of cerebellar cyclic GMP. Mice received one of the HCH isomers and were sacrificed for determination of cyclic GMP concentrations one hour later. Gamma-HCH increased cyclic GMP while alpha and delta-HCH decreased it. In addition, alpha and delta-HCH prevented the increase in cyclic GMP due to the gamma isomer. Picrotoxin increased cyclic GMP in a manner similar to that of gamma-HCH while strychnine produced only a small increase. All three HCH isomers inhibited the binding of 3H-TBOB (a ligand for the GABA-A-receptor linked chloride channel) in mouse cerebellum. It is concluded that the different HCH isomers can have different effects on cerebellar cyclic GMP accumulation and that these effects may be mediated through actions at the GABA-A receptor linked chloride channel.     

Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.     

Surfactant mediated enhanced biodegradation of hexachlorocyclohexane (HCH) isomers by Sphingomonas sp. NM05

Environmental biodegradation of several chlorinated pesticides is limited by their low solubility and sorption to soil surfaces. To mitigate this problem we quantified the effect of three biosurfactant viz., rhamnolipid, sophorolipid and trehalose-containing lipid on the dissolution, bioavailability, and biodegradation of HCH-isomers in liquid culture and in contaminated soil. The effect of biosurfactants was evaluated through the critical micelle concentration (CMC) value as determined for each isomer. The surfactant increased the solubilization of HCH isomers by 3-9 folds with rhamnolipid and sophorolipid being more effective and showing maximum solubilization of HCH isomers at 40 μg/mL, compared to trehalose-containing lipid showing peak solubilization at 60 μg/mL. The degradation of HCH isomers by Sphingomonas sp. NM05 in surfactant-amended liquid mineral salts medium showed 30% enhancement in 2 days as compared to degradation in 10 days in the absence of surfactant. HCH-spiked soil slurry incubated with surfactant also showed around 30-50% enhanced degradation of HCH which was comparable to the corresponding batch culture experiments. Among the three surfactants, sophorolipid offered highest solubilization and enhanced degradation of HCH isomers both in liquid medium and soil culture. The results of this study suggest the effectiveness of surfactants in improving HCH degradation by increased bioaccessibility.     

Kinetic and structural characterization of urease active site variants

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.     

Kinetic analysis of the coordinated interaction of SgrAI restriction endonuclease with different DNA targets

SgrAI restriction endonuclease cooperatively interacts and cleaves two target sites that include both the canonical sites, CPuCCGGPyG, and the secondary sites, CPuCCGGPy(A/T/C). It has been observed that the cleaved canonical sites stimulate SgrAI cleavage at the secondary sites. Equilibrium binding studies show that SgrAI binds to its canonical sites with a high affinity (Ka = 4-8 x 10(10) M-1) and that it has a 15-fold lower affinity for the cleaved canonical sites and a 30-fold lower affinity for the secondary sites. Steady-state kinetics reveals substrate cooperativity for SgrAI cleavage on both canonical and secondary sites. The specificity of SgrAI for the secondary site CACCGGCT, as measured by kcat/K is about 500-fold lower than that for the canonical site CACCGGCG, but this difference is reduced to 10-fold in the presence of the cleaved canonical sites. The efficiency of canonical site cleavage also increases by 3-fold when the cleaved canonical sites are present in the reaction. Furthermore, the substrate cooperativity for SgrAI cleavage is abolished for both types of sites in the presence of cleaved canonical sites. These results indicate that target site cleavage occurs via a coordinated interaction of two SgrAI protein subunits, where the subunit bound to the cleaved site stimulates the cleavage of the uncut site bound by the other subunit. The free subunits of SgrAI have the flexibility to bind different target sites and, consequently, assemble into various catalytically active complexes, which differ in their catalytic efficiencies.     

Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization.     

Enantioselective transformation of alpha-hexachlorocyclohexane by the dehydrochlorinases LinA1 and LinA2 from the soil bacterium Sphingomonas paucimobilis B90A

Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral alpha-hexachlorocyclohexane (alpha-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (-) enantiomer. Concurrent formation and subsequent dissipation of beta-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral alpha-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of alpha-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.